Sodalis glossinidlus is a facultative intracellular bacterium that is a secondary symbiont of the tsetse fly, which carries the trypanosome that causes African sleeping sickness. Pure culture techniques for growing the bacterium have only recently been published and, thus, procedures and techniques optimized for molecular genetic manipulation of Sodalis have not fully been developed. Furthermore, the physiological processes that enable this facultative endosymbiotic bacterium to survive and multiply within the insect, including the intracellular environment, are just beginning to be examined. Investigating these processes will contribute to the long-term scientific goals of the PI's research, which are to elucidate the physiological processes that are important for pathogens and non-pathogens to live within eukaryotic cells. The scientific goals of this project are to develop and refine molecular genetic tools for the purpose of testing hypotheses which address the contribution of two Sodalis putative iron acquisition systems to growth of Sodalis within insect cells and the tsetse fly. These systems are predicted to encode a protein that facilitates efficient utilization of heme as an iron source (HemS) and the achomobactin siderophore system. In Specific Aim 1, intron-based mutagenesis procedures will be optimized in Sodalis to generate directed mutations in hemS. The achromobactin genes will be eliminated from Sodalis by removing the plasmid that contains these genes. In specific aim 2, the ability of the Sodalis hemS and achromobactin mutants to grow in tsetse flies and in insect culture will be examined to determine which Sodalis iron acquisition genes are important for growth. In Specific Aim 3, the function of the putative Sodalis hemS and achromobactin genes will be verified by using the Sodalis genes to complement Shigella dysenteriae and E. coli strains that carry mutations in similar genes. In Specific Aim 4, the expression pattern of the Sodalis hemS and achromobactin genes will be examined when Sodalis is in the tsetse fly. PUBLIC HEALTH RELEVANCE: Studying the tsetse fly-Sodalis symbiosis will have significance to public health because the presence of Sodalis in the tsetse fly may increase tsetse-mediated transmission of the trypanosome that causes African sleeping sickness;thus, an increased understanding of the nature of the intracellular symbiosis of Sodalis and tsetse may yield information to help control tsetse-borne disease. Furthermore, many important human pathogens can live as endosymbionts and require high affinity iron transport for pathogenesis, and the data obtained from the work described in this proposal will provide information relevant to such pathogenic symbioses.